Summary of work: One theory of the mechanism of aging of post-mitotic tissues proposes that an accumulation of oxidative damage to cell components leads to a decline in cell function and ultimately death. The major source of oxidative stress, in the form of oxygen radicals, is mitochondrial respiration and one particularly provocative version of this theory of aging suggests that oxidative damage to mitochondrial DNA (mt-DNA) gives rise to deletions and other mutations, which accumulate during the many cycles of mitochondrial division that occur during the lifetime of a myocyte or neurone, eventually giving rise to a failure in mitochondrial energy transduction. In a collaboration with R.M. Anson and Dr. M. Dizdaroglu, mt-DNA isolated from the liver of young adult (6 mo) and senescent (24 mo) male rats was analyzed for oxidized base products using gas chromatography/mass spectrometry. In distinction to the literature, no generalized increase in oxidation products and, specifically, no increase in 8-oxo dG was found with aging. Nor was mt-DNA found to be more oxidized than nuclear DNA from the same animal. These studies are being extended by inclusion of more animals. This year we have also initiated a study on the response to aging and to oxidant stress of a novel mitochondrial endonuclease which is specific for 8-oxo dG adducts in DNA and which has been characterized by Deborah Croteau of LMG. In a prior collaboration with Ms. Croteau, this enzyme activity was found to be elevated in extracts of rat heart mitochondria as a function of senescence. This year we have shown this to be true also for liver mitochondria and have begun to test the hypothesis that this reflects an adaptive response to chronic oxidative stress. In a separate study, we have begun to assess the physiological significance of the age-linked accumulation of deletions in mt-DNA. To examine this on a cell-by-cell basis, we are taking advantage of FACS technology to separate human T- lymphocytes on the basis of mitochondrial membrane potential; we can then probe the different populations for deletions using PCR. We have demonstrated a greater fraction of cells with low mitochondrial membrane potential, and thus impaired mitochondrial functioning, in T-cells from elderly donors.